Methods of use of recombinant vasoactive protein from salivary gland of the black fly

ABSTRACT

The invention is drawn to vasodilative proteins from the salivary glands of the species, Simulium. The protein additionally has immunomodulating activities. Methods for recombinant production of the protein as well as biomedical uses are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. patent applicationSer. No. 09/702,647, filed Oct. 31, 2000, now issued as U.S. Pat. No.6,500,420, which is a divisional application of U.S. patent applicationSer. No. 09/036,355 filed Mar. 6, 1998, now issued as U.S. Pat. No.6,162,785, which claims the benefit of U.S. Provisional Application No.60/040,418, filed Mar. 13, 1997.

FIELD OF THE INVENTION

The invention relates to the production of recombinant proteins andtheir use in biomedical therapies.

BACKGROUND OF THE INVENTION

Hypertension is the most common cardiovascular disease. Many people inthe United States suffer from what is commonly referred to as “highblood pressure.” That is, have a systolic and/or diastolic bloodpressure above 140/90.

Elevated arterial pressure causes pathological changes in thevasculatury and hypertrophy of the left ventricle. As a consequence,hypertension has many deleterious effects on the body. For example, itis the principal cause of stroke, leads to disease of the coronaryarteries with myocardial infarction and sudden cardiac death, and is amajor contributor to cardiac failure, renal insufficiency, anddissecting aneurism of the aorta.

Pharmacological treatment of patients with high blood pressure willreduce morbidity, disability, and mortality from cardiovascular disease.Effective antihypertensive therapy will almost completely preventhemorrhagic strokes, cardiac failure, and renal insufficiency due tohypertension. Overall, there is a marked reduction in total strokes.

Antihypertensive drugs can be classified according to their sites ormechanisms of action. Arterial pressure is the product of cardiac outputand peripheral vascular resistance. Thus, such pressure can be loweredby actions of drugs on either the peripheral resistance or the cardiacoutput, or both. Drugs may reduce the cardiac output by eitherinhibiting myocardial contractility or decreasing ventricular fillingpressure. Reduction in ventricular filling pressure may be achieved byactions on the venous tone or on blood volume via renal effects. Drugscan reduce peripheral resistance by acting on smooth muscle to causerelaxation of resistance vessels or by interfering with the activity ofsystems that produce constriction of resistance vessels.

Vasodilators are a class of drugs which are commonly employed in thetherapy of heart failure, high blood pressure, and other variousconditions characterized by constricted blood vessels. Such conditionsinclude Raynaud's syndrome, certain post-surgical complications of brainsurgery involving subarachnoid hemorrhage, heart failure, anginapectoris, and hypertension.

Proteins from biting insects, particularly blood-feeding arthropods,have been shown to contain numerous pharmacologically-active substances,including vasodilating substances. The saliva from such insects containsuch substances to counteract many of the host's hemostatic defenses.Among these secretions are the potent vasodilating substances thatheighten blood flow to the feeding site.

The salivary components responsible for vasodilation are extremelyvaried as revealed by the recent characterization of purified factorsfrom several genera. Of several species of ticks analyzed, the saliva ofeach contained a lipid-derived prostaglandin that could account forvasodilative activity. Further, vasodilators play a role inskin-associated immune response.

Specific immunity has evolved as a sophisticated defense mechanism ofhigher organisms. In humans, cell-mediated immunity and humoral immunityare the two major mechanisms. Both of these responses have a high levelof specificity directed to antigenic epitopes expressed on molecularcomponents of foreign agents.

There are several clinical settings where it is desirable to suppress animmune response. These situations include organ transplantation,treatment of autoimmune disorders, and prevention of Rh hemolyticdisease of the newborn.

Because of the importance of providing hypertension therapies, potentvasodilators are needed. Additionally, agents which are capable ofmodulating the immune response and aiding in wound healing areadditionally desirable.

SUMMARY OF THE INVENTION

Purified vasoactive proteins from the salivary glands of theblood-feeding black fly, Simulium sp. are provided. The proteins finduse in biomedical therapies, particularly where peripheral resistanceand stenoses are problems. The proteins are also useful as regulators ofthe immune response and as promoters of wound healing.

The nucleotide sequence encoding the proteins, as well as methods forproducing recombinant protein, are additionally provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the presence of erythema in NZW rabbits followingintradermal injection of SGE of female S. vittatum or rSVEP.

DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for use as therapeutic vasodilating agents,i.e., as regulators of blood pressure are provided. The agents can beused to regulate blood flow to a wound site promoting wound healing.Additionally, the compositions of the invention can be utilized tomodulate the immune response.

The compositions of the invention comprise vasoactive proteins from thesalivary glands of the blood-feeding black fly. The proteins exhibitvasodilative activity and wound healing promoting properties, as well asthe capacity to suppress certain immune responses in a mammal.Substantially purified preparations of the proteins are provided. Suchsubstantially purified preparations include protein substantially freeof any compound normally associated with the protein in its naturalstate. Such proteins can be assessed for purity by SDS-PAGE,chromatography, electrophoresis or other methods known in the art. See,M. P. Deutscher (ed.), Guide to Protein Purification, Academic Press,Inc. (1990).

The terms substantially pure or substantially purified are not meant toexclude artificial or synthetic mixtures of the protein with othercompounds. It is recognized that the vasoactive proteins of the presentinvention include those proteins homologous to, and having essentiallythe same biological properties as, the vasoactive protein describedherein, and particularly the protein disclosed herein in SEQ ID NO: 2.This definition is intended to encompass natural allelic variations inthe genes.

The invention additionally encompasses the nucleotide sequences, whichencode the proteins of the invention. The nucleotide sequence of thecoding sequence from S. vittatum is provided in SEQ ID NO: 1.Additionally, cloned genes of the present invention can be of otherspecies of origin. Thus, DNAs which hybridize to the nucleotide sequenceof the vasoactive gene from the black fly are also an aspect of thisinvention. Conditions, which will permit other DNAs to hybridize to theDNA disclosed herein, can be determined in accordance with knowntechniques. For example, hybridization of such sequences may be carriedout under conditions of reduced stringency, medium stringency or evenstringent conditions (e.g., conditions represented by a wash stringencyof 35-40% Formamide with 5× Denhardt's solution, 0.5% SDS and 1× SSPE at37° C.; conditions represented by a wash stringency of 40-45% Formamidewith 5× Denhardt's solution, 0.5% SDS, and 1× SSPE at 42° C.; andconditions represented by a wash stringency of 50% Formamide with 5×Denhardt's solution, 0.5% SS and 1× SSPE at 42° C., respectively, to DNAencoding the vasoactive genes disclosed herein in a standardhybridization assay. See J. Sambrook et al., Molecular Cloning, ALaboratory Manual (2d Ed. 1989) (Cold Spring Harbor Laboratory).

In general, sequences which code for the vasoactive protein andhybridize to the nucleotide sequence disclosed herein will be at least75% homologous, 85% homologous, and even 95% homologous or more with thesequences. Further, the amino acid sequences of the vasoactive proteinsisolated by hybridization to the DNA's disclosed herein are also anaspect of this invention. The degeneracy of the genetic code, whichallows different nucleic acid sequences to code for the same protein orpeptide, is well known in the literature. See, e.g., U.S. Pat. No.4,757,006.

The hybridization probes may be cDNA fragments or oligonucleotides, andmay be labeled with a detectable group as known in the art. Pairs ofprobes which will serve as PCR primers for the vasoactive gene or aprotein thereof may be used in accordance with the process described inU.S. Pat. Nos. 4,683,202 and 4,683,195.

It is recognized that the nucleotide and peptide sequences of theinvention may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the peptides and proteins can be prepared bymutations in the DNA. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Kunkel, T.(1985) Proc. Natl. Acad. Sci. USA 82:488-492; Kunkel et al. (1987)Methods in Enzymol. 154:367-382; U.S. Pat. No. 4,873,192; Walker andGaastra (eds.) Techniques in Molecular Biology, MacMillan PublishingCompany, N.Y. (1983) and the references cited therein. Thus, thenucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant forms. Likewise, the peptides andproteins of the invention encompass both naturally occurring andmodified forms thereof. Such variants will continue to possess thedesired activity. Obviously, the mutations that will be made in the DNAencoding the variant must not place the sequence out of reading frameand preferably will not create sequences deleterious to expression ofthe gene product. See, EP Patent Application Publication No. 75,444.

Thus proteins of the invention include the naturally occurring forms aswell as variants thereof. These variants will be substantiallyhomologous and functionally equivalent to the native protein. A variantof a native protein is “substantially homologous” to the native proteinwhen at least about 80%, more preferably at least about 90%, and mostpreferably at least about 95% of its amino acid sequence is identical tothe amino acid sequence of the native protein. A variant may differ byas few as 1, 2, 3, or 4 amino acids. By “functionally equivalent” isintended that the sequence of the variant defines a chain that producesa protein having substantially the same biological activity as thenative protein of interest. Such functionally equivalent variants thatcomprise substantial sequence variations are also encompassed by theinvention. Thus a functionally equivalent variant of the native proteinwill have a sufficient biological activity to be therapeutically useful.By “therapeutically useful” is intended effective in achieving atherapeutic goal as discussed in more detail below.

Methods are available in the art for determining functional equivalence.Biological activity can be measured using assays specifically designedfor measuring activity of the native protein, including assays describedin the present invention. Additionally, antibodies raised against thebiologically active native protein can be tested for their ability tobind to the functionally equivalent variant, where effective binding isindicative of a protein having conformation similar to that of thenative protein.

DNA sequences can also be synthesized chemically or modified bysite-directed mutagenesis to reflect the codon preference of the hostcell and increase the expression efficiency.

The proteins of the invention can be “engineered” in accordance with thepresent invention by chemical methods or molecular biology techniques.Molecular biology methods are most convenient since proteins can beengineered by manipulating the DNA sequences encoding them. Genomic DNA,cDNA, synthetic DNA, and any combination thereof may be used for thispurpose. Genomic DNA sequences or cDNA sequences encoding proteins canbe isolated based on the amino acid sequence of proteins or certainprotein properties. Many methods of sequence isolation are known in theart of molecular biology. See particularly Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (Cold Spring Harbor LaboratoryPress, Plainview, N.Y.), herein incorporated by reference.

Once the nucleotide sequences encoding the vasoactive proteins of theinvention have been isolated, they can be manipulated and used toexpress the protein in a variety of hosts including other organisms,including microorganisms.

Once the nucleotide sequence is identified and known, those skilled inthe art can produce large quantities of the protein for therapeutic use.Accordingly, recombinant protein and methods for producing therecombinant protein are encompassed by the present invention. In thismanner, the nucleotide sequence encoding the vasoactive protein can beutilized in vectors for expression in various types of host cells,including both procaryotes and eucaryotes, to produce large quantitiesof the protein, or active analogues, or fragments thereof, and otherconstructs capable of inducing vasodilation or temporarily suppress theimmune response in a mammal.

Generally, methods for the expression of recombinant DNA are known inthe art. See, for example, Sambrook et al. Molecular Cloning, ColdSpring Harbor Laboratory (1989). Additionally, host cells and expressionvectors, such as the baculovirus expression vector may be employed incarrying out the present invention, as described in U.S. Pat. Nos.4,745,051 and 4,879,236. In general, a baculovirus expression vectorcomprises a baculovirus genome containing the gene to be expressedinserted into the polyhedron gene at a position ranging from thepolyhedron transcriptional start signal to the ATG start site and underthe transcriptional control of a baculovirus polyhedron promoter.

A broad variety of suitable procaryotic and microbial vectors areavailable. Likewise, the promoters and other regulatory agents used inexpression of foreign proteins are available in the art. Promoterscommonly used in recombinant microbial expression vectors are known inthe art and include the beta-lictamase (penicillinase) and lactosepromoter systems (Chang et al. (1978) Nature, 275:615 and Goeddel et al.(1979) Nature, 281:544); A tryptophan (TRP) promoter system (Goeddel etal. (1980) Nucleic Acids Res., 8:4057 and the EPO ApplicationPublication No. 36,776); and the Tac promoter (DeBoer et al. (1983)Proc. Natl. Acad. Sci. USA, 80:21). While these are commonly used, othermicrobial promoters are available. Details concerning nucleotidesequences of many have been published, enabling a skilled worker tooperably ligate them to DNA encoding the protein in plasmid or viralvectors. See, for example, Siedenlist et al. (1980) Cell 20:269.

Eukaryotic microbes such as yeast may be transformed with suitableprotein-encoding vectors. See, e.g., U.S. Pat. No. 4,745,057.Saccharomyces cerevisiae is the most commonly used among lowereukaryotic host microorganisms, although a number of other strains arecommonly available. Yeast vectors may contain an origin of replicationfrom the 2 micron yeast plasmid or an autonomously replicating sequence(ARS), a promoter, DNA encoding the desired protein, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al. (1979) Nature, 282:9;Kingsman et al. (1979) Gene, 7:141; Tschemper et al. (1980) Gene,10:157). This plasmid contains the trp 1 gene, which provides aselection marker for a mutant strain of yeast lacking the ability togrow in tryptophan, for example ATCC No. 44076 or PEP4-1 (Jones,Genetics 85, 12 (1977)). The presence of the trp 1 lesion in the yeasthost cell genome then provides an effective environment for detectingtransformation by growth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, alcohol dehydrogenase, adenylate cyclase,3-phosphoglycerate kinase (Hitzeman et al. (1980) J. Biol. Chem.255:2073) and other glycolytic enzymes (Hess et al. (1968) J. Adv.Enzyme Reg., 7:149; and Holland et al. (1978) Biochemistry, 17:4900)such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase. Suitable vectorsand promoters for use in yeast expression are further described in R.Hitzeman et al. EPO Publication. No. 73,657.

The compositions of the present invention can be formulated intopharmaceutical preparations for therapeutic use. As a vasodilator, thecompositions find use for atherosclerosis of extremities, for heartfailure, for hypertension, for peripheral resistance, stenoses, and thelike, particularly peripheral vasodilation.

The compositions of the invention can also be used to temporarilysuppress the immune system. In this manner, a mammal can be desensitizedto the effects of an immunogen by parenteral administration of thevasoactive protein, active analogs or fragments thereof. For modulatingthe immune system, the proteins can be utilized to inhibit or preventthe development of antibodies or cellular immunity to a protein, totreat graft rejection, autoimmune diseases, and the like.

The compositions of the invention find use as promoters of woundhealing. Application to the wound site results in an increased rate ofhealing.

The compositions of the invention can be used alone or in combinationwith other vasoactive and therapeutic agents. Other agents are known inthe art.

The vasoactive compositions can be formulated according to known methodsto prepare pharmaceutically useful compositions, such as by admixturewith a pharmaceutically acceptable carrier vehicle. Suitable vehiclesand their formulation are described, for example, in Remington'sPharmaceutical Sciences 16th ed., Osol, A. (ed.), Mack Easton Pa.(1980). In order to form a pharmaceutically acceptable compositionsuitable for effective administration, such compositions will contain aneffective amount of the vasoactive protein, either alone, or with asuitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control theduration of action. Controlled release preparations may be achieved bythe use of polymers to complex or absorb the antiviral compositions. Thecontrolled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinylpyrrolidone, ethylene-vinylacetate, methylcellulose,carbosymethylcellulose, or protamine sulfate). The rate of drug releasemay also be controlled by altering the concentration of suchmacromolecules.

Another possible method for controlling the duration of action comprisesincorporating the therapeutic agents into particles of a polymericsubstance such as polyesters, polyamiono acids, hydrogels, poly(lacticacid) or ethylene vinylacetate copolymers. Alternatively, it is possibleto entrap the therapeutic agents in microcapsules prepared, for example,by coacervation techniques or by interfacial polymerization, forexample, by the use of hydroxymethyl cellulose or gelatin-microcapsulesor poly(methylmethacrylate) microcapsules, respectively, or in a colloiddrug delivery system, for example, liposomes, albumin, microspheres,microemulsions, nanoparticles, nanocapsules, or in macroemulsions. Suchteachings are disclosed in Remington's Pharmaceutical Sciences (1980).

It is contemplated that the inhibitory compositions of the presentinvention will be administered by an individual in therapeuticallyeffective amounts. That is, in an amount sufficient to regulate bloodpressure and/or suppress the immune response. The effective amount ofthe composition will vary according to the weight, sex, age, and medicalhistory of the individual. Other factors which influence the effectiveamount may include, but are not limited to, the severity of thepatient's condition, the stability of the vasoactive protein, thekinetics of interaction in the recipient, previous exposure to thevasoactive protein, kidney or other disease, etc. Typically, for a humansubject, an effective amount will range from about 0.1 ng to about 100mg, specifically, from about 1 ng to about 10 mg, more specifically fromabout 10 ng to about 1 mg.

The pharmaceutically prepared inhibitory compositions of the inventionmay be provided to a patient by means will known in the art. Such meansof introduction include oral means, intranasal means, subcutaneousmeans, intramuscular means, topical, intradermal means, intravenousmeans, intraarterial means, or parenteral means.

The vasoactive proteins of the present invention may be dissolved in anyphysiologically tolerated liquid in order to prepare an injectablebolus. It is generally preferable to prepare such a bolus by dissolvingthe molecule in normal saline.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL

Many modifications and other embodiments of the invention will come tomind in one skilled in the art to which this invention pertains havingthe benefit of the teachings presented in the foregoing descriptions andthe associated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed.Although specific terms are employed, they are used in a generic anddescriptive sense only and not for purposes of limitation, and thatmodifications and embodiments are intended to be included within thescope of the appended claims.

Salivary gland extracts of several Simulium were shown to containvasodilative activity as measured by the rapid and persistent inductionof erythema in response to intradermal injection into rabbit skin. Testsfor physical stability of the activities indicated that the vasodilatorswere proteinaceous and heat stable. The electrospray ionization massspectroscopy of the S. vittatum protein detected a mass of about 15,000Daltons.

Methods for the construction of S. vittatum salivary gland cDNA libraryand cloning of specific cDNA of Simulium vittatum salivary glanderythema protein (SVEP) was performed by the following steps:

1. SVEP was purified from salivary glands and sent to the HarvardMicrochemistry Laboratory where it was subjected to limited digestionwith trypsin. Two peptides, CT29 (SEQ ID NO: 3) and CT51 (SEQ ID NO: 4)were sequenced by an automated Edman degradation procedure.

2. Messenger RNA (mRNA) was isolated from SGE of S. vittatum. Acommercially-available kit was used to prepare the cDNA library (ZAPEXPRESS™ cDNA synthesis kit, Strategene, La Jolla, Calif.).

3. A fragment of the SVEP cDNA was generated by PCR using degenerateprimers that were designed based on knowledge of the partial amino acidsequence revealed in sequencing of the purified protein. A commerciallyavailable kit (TA Cloning® System, Invitrogen) was used to clone the PCRproduct. Sequencing of the cDNA and comparison of the translated aminoacid sequence confirmed the validity of the clone. Further, the relativeorder of the two peptides and the intervening amino acids weredetermined (SEQ ID NO: 5). A digoxigenin-labeled (DIG) probe wasgenerated for use in screening the cDNA library to recover thefull-length clone.

4. Screening of the library produced a full-length clone that providedthe remaining codes for all the amino acids, including a hydrophobicleader sequence that is cleaved from the mature, functional protein.Analysis of the remaining bases revealed that the mRNA for this proteinhas a relative small number of non-translated base sequences at the Nand C termini (SEQ ID NO: 1).

5. Calculation of the putative molecular weight of the mature proteinwhich would be generated by the cDNA clone was 15,348.9 Daltons, whichis 1.23 to 2.58 Daltons less than the weight of the HPLC purifiedprotein determined by ESIMS.

C. Production of Recombinant SVEP Protein (rSVEP) Via BaculovirusExpression System

1. A commercially available baculovirus vector (pBacPAK8, ClontechLaboratories, Inc., Palo Alto, Calif.) and cDNA of SVEP were digestedwith PstI and XhoI restriction enzymes and religated to form arecombinant plasmid.

2. Recombinant virus was produced by co-infection of Sf9 cells with thepBacPAK8/SVEP and baculovirus DNA digested with BSU36I.

3. Recombinant virus was purified by plaque assay and amplified.

4. Production of SVEP DNA in the recombinant virus was confirmed by PCRamplification of cellular DNA isolated from infected cultures.

5. Synthesis and secretion of protein of the appropriate molecularweight was demonstrated in SDS/PAGE of proteins present in the cellularcultures of recombinant-virus infected cells and absent from cellularsupernatants of wild-type virus infected cells.

D. Quantitative Analysis of rSVEP

1. By examination of silver-stained, SDS protein gels, it was determinedthat rSVEP was ≧90% of the total protein in cell culture supernatants at48 hr post infection.

2. Total protein concentration was determined using the Lowry method.Based on the observations of #1 above, rSVEP protein concentration wasestimated as the difference between total protein concentration incellular supernatants of BV/SVEP infected cells and wild-type infectedcells.

3. Using these quantitative measurements, the potency of rSVEP wasestimated by bioassay in rabbit skin as described previously. The limitof detectable erythema following injection was approximately 1 ng, andwas equivalent to the activity present in 0.017 pairs of S. vittatumsalivary glands. For a protein of molecular weight 15,315 Daltons, thisis equivalent to 65 femtomoles (FIG. 1).

E. Physical Properties of rSVEP

1. Native and recombinant SVEP have a compact tertiary structure thatcauses the protein to migrate at a faster rate, when subjected to gelsieving techniques, than would be predicted by molecular weight alone.Treatment with the disulfide reducing reagent, 2-mercaptoethanol, delaysmobility and thus indicates that the two cysteines form a disulfidebond. Because these two amino acids are located at the two differentends of the sequence, substantial folding of the protein must occur toaccommodate bond formation.

2. Amino acid composition of SVEP shows a relative high percentage ofbasic amino acids (see SEQ ID NO: 2; lysine, coded as K, and arginine,coded as R). Based on TSK gel sieving and protein staining patterns itis likely that the folded protein displays these basic moieties on itssurface to produce a positively charged molecule.

F. Therapeutic Uses of rSVEP

1. Test of rSVEP efficacy in facilitating wound healing.

a. Using NZW rabbits, sterile, surgical open and closed wounds will becreated. rSVEP or control solution will be injected intradermally orsubcutaneously on a daily basis.

b. Differentiation of vasoactive effects from inflammation will bedetermined using 1) laser doppler imagery for reperfusion, 2)histopathological evaluation for granulation tissue and 3) measurementof inflammatory cytokines, I11α, I11β and TNFα.

c. The rate of healing will be determined using 3 measures: Planimetryto determine rate of open wound healing, histological evaluation todetermine progression from inflammatory stage to repair stage, andtensiometry to determine strength of tissue repair.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

                   #             SEQUENCE LISTING<160> NUMBER OF SEQ ID NOS: 5 <210> SEQ ID NO 1 <211> LENGTH: 548<212> TYPE: DNA <213> ORGANISM: Simulium vittatum <220> FEATURE:<221> NAME/KEY: CDS <222> LOCATION: (49)...(507) <400> SEQUENCE: 1ctgaagtgta aagtacttaa atcattcggt gggaattatc cagcaagt atg #agc atc      57                    #                  #                 Met # Ser Ile                    #                  #                   #1 aca caa agc ttc ttt gtt tta acc ctt gcc at#a ttt ggt gct gca tca      105Thr Gln Ser Phe Phe Val Leu Thr Leu Ala Il #e Phe Gly Ala Ala Ser     5              #      10             #      15gac aac cca att gct gat aga aaa tgt atc gt#c atc agt gac ggg gac      153Asp Asn Pro Ile Ala Asp Arg Lys Cys Ile Va #l Ile Ser Asp Gly Asp 20                  # 25                  # 30                  # 35ctg gtt atg cac gag cga aaa ccc ggt caa ga#g ttc cca tac tat gtc      201Leu Val Met His Glu Arg Lys Pro Gly Gln Gl #u Phe Pro Tyr Tyr Val                 40  #                 45  #                 50tac atg atc ccg aag ggt aca gag tac gac ga#t caa cga tgg atc ctg      249Tyr Met Ile Pro Lys Gly Thr Glu Tyr Asp As #p Gln Arg Trp Ile Leu             55      #             60      #             65gag agt gtg gga gga gat cac tat aag ctg aa#g aac aag ttt tcc gga      297Glu Ser Val Gly Gly Asp His Tyr Lys Leu Ly #s Asn Lys Phe Ser Gly         70          #         75          #         80cgg tat ttg gtg tat ggc acc ttt gat tat tt#c ctc acg gca gga gca      345Arg Tyr Leu Val Tyr Gly Thr Phe Asp Tyr Ph #e Leu Thr Ala Gly Ala     85              #     90              #     95gcc gtc aga gag atg gat cat ttc aaa ttc ac#t gct gat ggg acg ggc      393Ala Val Arg Glu Met Asp His Phe Lys Phe Th #r Ala Asp Gly Thr Gly100                 1 #05                 1 #10                 1 #15aag tat gac atc tct agc aaa gcg aat ggt ca#t cct cga tct cgc ggc      441Lys Tyr Asp Ile Ser Ser Lys Ala Asn Gly Hi #s Pro Arg Ser Arg Gly                120   #               125   #               130aaa aat tgg gga gtc atg aaa gat ggt gag aa#g cac tat ttc act gtt      489Lys Asn Trp Gly Val Met Lys Asp Gly Glu Ly #s His Tyr Phe Thr Val            135       #           140       #           145gaa aat tgt cag gaa taa taaataagaa atgttgaagt tg#aaaaaaaa             537 Glu Asn Cys Gln Glu  *         150aaaaaaaaaa a                #                   #                  #      548 <210> SEQ ID NO 2 <211> LENGTH: 152 <212> TYPE: PRT<213> ORGANISM: Simulium vittatum <400> SEQUENCE: 2Met Ser Ile Thr Gln Ser Phe Phe Val Leu Th #r Leu Ala Ile Phe Gly 1               5   #                10   #                15Ala Ala Ser Asp Asn Pro Ile Ala Asp Arg Ly #s Cys Ile Val Ile Ser            20       #            25       #            30Asp Gly Asp Leu Val Met His Glu Arg Lys Pr #o Gly Gln Glu Phe Pro        35           #        40           #        45Tyr Tyr Val Tyr Met Ile Pro Lys Gly Thr Gl #u Tyr Asp Asp Gln Arg    50               #    55               #    60Trp Ile Leu Glu Ser Val Gly Gly Asp His Ty #r Lys Leu Lys Asn Lys65                   #70                   #75                   #80Phe Ser Gly Arg Tyr Leu Val Tyr Gly Thr Ph #e Asp Tyr Phe Leu Thr                85   #                90   #                95Ala Gly Ala Ala Val Arg Glu Met Asp His Ph #e Lys Phe Thr Ala Asp            100       #           105       #           110Gly Thr Gly Lys Tyr Asp Ile Ser Ser Lys Al #a Asn Gly His Pro Arg        115           #       120           #       125Ser Arg Gly Lys Asn Trp Gly Val Met Lys As #p Gly Glu Lys His Tyr    130               #   135               #   140Phe Thr Val Glu Asn Cys Gln Glu 145                 1 #50<210> SEQ ID NO 3 <211> LENGTH: 22 <212> TYPE: PRT<213> ORGANISM: Simulium vittatum <400> SEQUENCE: 3Gly Lys Asn Trp Gly Val Met Lys Asp Gly Gl #u Lys His Tyr Phe Thr 1               5   #                10   #                15Val Glu Asn Cys Gln Glu             20 <210> SEQ ID NO 4<211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Simulium vittatum<400> SEQUENCE: 4 Lys Pro Gly Gln Glu Phe Pro Tyr Tyr Val Ty#r Met Ile Pro Lys  1               5   #                10  #                15 <210> SEQ ID NO 5 <211> LENGTH: 109 <212> TYPE: PRT<213> ORGANISM: Simulium vittatum <400> SEQUENCE: 5Lys Pro Gly Gln Glu Phe Pro Tyr Tyr Val Ty #r Met Ile Pro Lys Gly 1               5   #                10   #                15Thr Glu Tyr Asp Asp Gln Arg Trp Ile Leu Gl #u Ser Val Gly Gly Asp            20       #            25       #            30His Tyr Lys Leu Lys Asn Lys Phe Ser Gly Ar #g Tyr Leu Val Tyr Gly        35           #        40           #        45Thr Phe Asp Tyr Phe Leu Thr Ala Gly Ala Al #a Val Arg Glu Met Asp    50               #    55               #    60His Phe Lys Phe Thr Ala Asp Gly Thr Gly Ly #s Tyr Asp Ile Ser Ser65                   #70                   #75                   #80Lys Ala Asn Gly His Pro Arg Ser Arg Gly Ly #s Asn Trp Gly Val Met                85   #                90   #                95Lys Asp Gly Glu Lys His Tyr Phe Thr Val Gl #u Asn Cys            100       #           105

That which is claimed is:
 1. A method for lowering peripheral vascularresistance in a mammal, said method comprising administering atherapeutically effective amount of a polypeptide comprising the aminoacid sequence set forth in SEQ ID NO: 2, that increases blood flowwhereby the peripheral vascular resistance is lowered.
 2. A method forlowering peripheral vascular resistance in a mammal, said methodcomprising administering a therapeutically effective amount of apolypeptide comprising the mature form of the amino acid sequence of SEQID NO:2 that increases blood flow, whereby the peripheral vascularresistance is lowered.
 3. The method of claim 1, wherein saidpolypeptide is produced by recombinant methods.
 4. A method for loweringperipheral vascular resistance in a mammal, said method comprisingadministering a therapeutically effective amount of a polypeptideencoded by a nucleotide sequence comprising the sequence set forth innucleotides 49 504 of SEQ ID NO:1 that increases blood flow, whereby theperipheral vascular resistance is lowered.
 5. The method of claim 1,wherein said step of administering said polypeptide comprisesadministration by intradermal injection.